[Association between short-term exposure to ambient nitrogen dioxide and the risk of heart failure hospitalization].

Objective: To assess the association between short-term exposure level of nitrogen dioxide and the hospitalization risk of heart failure. Methods: Based on China-PEACE Retrospective Heart Failure Study, 117 364 hospitalized patients with heart failure were recruited from 92 hospitals in 62 cities throughout China between January 1, 2015 and December 31, 2015. The daily exposure level of nitrogen dioxide, temperature, and humidity in the same cities during the same period were also collected. We applied the generalized additive model and Bayesian hierarchical model to quantify the lagged effect and cumulative effect of short-term (0-3 days) exposure to ambient nitrogen dioxide on the hospitalization risk of heart failure. We further conducted stratified analyses by age, region, and season to identify any difference in the associations between short-term nitrogen dioxide exposure and heart failure among subgroups. Results: The mean age for participants in the analysis was (70.32±12.22) years. The median, minimum and maximum of daily nitrogen dioxide concentration in 62 cities from January 1, 2015 to December 31, 2015 was 26.4 µg/m3, 2.33 µg/m3 and 150.25 µg/m3, respectively. The exposure level of nitrogen dioxide at the same day was associated with the hospitalization risk of heart failure (OR=1.022, 95%CI: 1.012, 1.031). Significant effects were also observed in the moving average concentrations from lag 0-1 to lag 0-3 day (OR=1.020, 95%CI: 1.009, 1.030; OR=1.016, 95%CI: 1.004, 1.028; OR=1.013, 95%CI: 1.001, 1.026). Moreover, all of the associations between short-term exposure to nitrogen dioxide and the risk of heart failure hospitalization were statistically significant, with no significant difference in all subgroups stratified by age, region, and season. Conclusion: A higher level of short-term exposure to nitrogen dioxide could trigger more hospitalizations with heart failure.

[Correlation study of Staphylococcus aureus superantigens and formation of human ear keloid].

Objective: To investigate the correlation of Staphylococcus aureus superantigens and human ear keloid formation. Methods: The retrospective case-control study method was used. The discarded keloid tissue was collected from 10 patients (9 females and 1 male, aged 19-59 years) with ear keloid after core excision of ear keloid, and the discarded normal skin tissue was collected from 3 female patients (aged 20-24 years) with pigmented nevus after operation, who were admitted to affiliated Hospital of Nantong University from June 2017 to March 2018. The exudation from ear keloid surface was collected for bacteria culture and identification. The keloid and normal skin tissue were collected to detect the protein expression of Staphylococcus aureus enterotoxin A+enterotoxin B+toxic shock syndrome toxin-1 (TSST-1) by Western blotting, and the keloid was divided into superantigen positive group and superantigen negative group according to the protein expression of Staphylococcus aureus enterotoxin A+enterotoxin B+TSST-1. The protein expressions of T cell receptor (TCR) V ß of keloid in 2 groups were detected by Western blotting. Collagen fibers formation and inflammatory cells infiltration in keloid dermis of 2 groups were observed by Masson and hematoxylin eosin staining, respectively. Enzyme-linked immunosorbent assay method was used to detect expression of Staphylococcus aureus enterotoxin A, enterotoxin B, and TSST-1 in keloids with superantigen positive. Data were statistically analyzed with paired sample t test. Results: Bacteria were cultured from the exudation on ear keloid surface, hemolysis was observed around the dominant bacteria after being cultured for 24 hours, and the colony was white or golden yellow, which was identified as Staphylococcus aureus. The protein expression of Staphylococcus aureus enterotoxin A+enterotoxin B+TSST-1 of normal skin was negative in 3 patients, and the protein expression was 0.267±0.016. The protein expression of Staphylococcus aureus enterotoxin A+enterotoxin B+TSST-1 of keloid was positive in 4 patients, with the protein expression of 0.472±0.016, which was included into superantigen positive group, and the protein expression of Staphylococcus aureus enterotoxin A+enterotoxin B+TSST-1 of keloid was negative in 6 patients, with the protein expression of 0.255±0.004, which was included into superantigen negative group. The protein expression of Staphylococcus aureus enterotoxin A+enterotoxin B+TSST-1 of keloid in superantigen positive group was obviously higher than that of keloid in superantigen negative group and that in normal skin (t=15.22, 8.63, P＜0.01). The protein expression of TCR Vß in keloid of superantigen positive group was 0.389±0.023, which was significantly higher than 0.169±0.014 in superantigen negative group (t=8.62, P＜0.01). Masson staining showed that a large number of collagen fibers existed in dermis of keloid in 2 groups. HE staining showed that there were a small number of inflammatory cells infiltration around the blood vessels of keloid dermis in superantigen negative group and a large number of inflammatory cells infiltration around the blood vessels of keloid in superantigen positive group. Among the 4 patients with superantigen positive keloid, Staphylococcus aureus enterotoxin A was positive in 2 patients, Staphylococcus aureus enterotoxin B was positive in 2 patients, and among that Staphylococcus aureus enterotoxin A and enterotoxin B were both positive in 1 patient, with no TSST-1 detected in any patient. Conclusions: The superantigen secreted by Staphylococcus aureus is one of the many causes of ear keloid, which may be related to the activation of keloid signal pathway by superantigen of Staphylococcus aureus.

[A comparison of clinical characteristics between patients with and without"recurrence"RNA positive with COVID-19].

Objective: To analysis the clinical characteristics of"recurrence"RNA positive patients with Coronavirus disease 2019 （COVID-19） and compared with those without"recurrence". Methods: 98 patients with COVID-19 in Wuhan Jinyintan Hospital and designated treatment hospitals in Quanzhou were included in this study from February 2020 to April 2020. There were 55 males and 43 females， aged from15 to 83 years， with a median age of 57.5 years， in which 20 cases were complicated with basic diseases. 15 of these patients had been diagnosed and hospitalized had been found as"recurrence"2019-nCoV RNA positive after discharge while the other 83 cases were all negative. The clinical classification of all patients was common type. Clinical data of the COVID-19 RNA"recurrence"patients were collected， and general situations， symptoms， laboratory examinations and CT images were also observed and analyzed. The patients were divided into 2019-nCoV"recurrent"group and 2019-nCoV"non-recurrent"group. There are 10 males and 5 females in 2019-nCoV"recurrent"group while 45 males and 38 females in"non-recurrent"group （χ²=0.800，P=0.371）. The age of 2019-nCoV"recurrent"group （57±21） was higher than that of"non-recurrent"group（53±17）. 8 of 15 the COVID-19"recurrent"group patients and 12 of 83"non-recurrent"patients have basic diseases. IgG and IgM of 2019-nCoV， IL-6， procalcitonin， ESR， CRP， BNP and other serum biochemical index levels were measured and compared between groups. Results: （1） The proportion of patients with common type of COVID-19 was 15.3% during 2-week medical observation after discharge. （2） All of the 2019-nCoV"recurrent"patients were hospitalized due to COVID-19 RNA positive， when they were quarantined after discharged from hospital. All the patients with mild symptoms which were clarified as common type， including 5 cases of fever， 6 cases of cough， 5 cases of expectoration， and 2 cases of slight shortness of breath. The time of symptoms appeared on （5.73±2.82） days after discharge. （3） The serum procalcitonin of all 2019-nCoV"recurrent"group patients were normal（all<0.05 ng/ml）. The BNP of"recurrent"group （151±171） ng/L， was higher than that of"non-recurrent"group （63±78） ng/L （t = 3.207， P = 0.000）. There was no significant difference in laboratory tests like leukocyte ［（6.17±2.4） and （6.04±2.41）×109/L］， lymphocyte［（1.59±0.52） and （1.32±0.64）×109/L］， CRP ［（12.54±28.20） and （21.74±25.63）mg/L］， ESR ［（31.07±28.72） and （34.10±22.16）mm/1 h］， AST ［（24.73±9.15） and （30.24±23.20）U/L］， ALT ［（22.60±12.82） and （36.47±34.12）U/L）， LDH ［（268±208） and （270±164）U/L］， D-dimer ［（0.60±0.50） and （0.84±0.98）µg/L］， ferritin ［（294±195） and （395±319）µg/L］， IL-6 ［（9.17±6.42） and （14.28±17.74）ng/Lï¼½ and BUN （5.77±2.66） and （4.74±2.81）U/Lï¼½ between"recurrent"and"non-recurrent"groups （all P>0.05）. （4） In"recurrent"group， ground glass， exudative or solid lesions could be found in most of the chest CT performed on re-admission. Meanwhile， fibrosis lesion was relatively rare. （5） There were no secondary transmissions were found to be caused by the 2019-nCoV"recurrent"group patients. Conclusions: Most of the 2019-nCoV patients had underlying diseases and active lesions were still found in CT images， so the possibility of virus replication may still exist. All"recurrent"patients had mild illness which may suggest that they were in recovery stage, and no evidence of transmission is found.

Ultrastrong and Stiff Carbon Nanotube/Aluminum-Copper Nanocomposite via Enhancing Friction between Carbon Nanotubes.

Researchers have been aiming to replace copper with carbon nanotube/copper nanocomposites, which are lighter and exhibit better electrical, mechanical, and thermal properties. However, the strength is far below pure carbon nanotube assembly and even much lower than some copper-based alloys. This disadvantage hinders the extensive application of carbon nanotube/copper nanocomposites. In this study, the carbon nanotube/aluminum-copper nanocomposites with ultra-strength and stiffness were prepared. The strength and elasticity modulus of composite reached as high as 6.6 and 500 GPa, respectively, while a high conductivity of 1.8 × 107 S/m was maintained. This can be attributed to the diffusion of Cu and Al atoms into the carbon nanotube fiber, which enhances friction between the carbon nanotubes by "pinning" and "bridging". This structure provides us with novel insights into the design of carbon nanotubes/metal nanocomposites with ultrahigh strength and conductivity.

Development of Individualized Induced Pluripotent Stem Cells From Fibroblasts of Keloid Lesions in Patients.

OBJECTIVE: Presently, interesting research related to induced pluripotent stem cells (iPSCs) is emerging. However, the development of new therapies and techniques for treatment of refractory diseases is still required in dermatology. We are exploring novel methods to provide stem cell therapy and elucidate research mechanisms underlying troublesome diseases by reprogramming iPSCs from the fibroblasts of keloid lesions from patients in vitro. METHOD: Here, we identified the expression of fibroblastic genes in the fibroblast derived from diseased individuals. Corresponding iPSCs were then produced by transfecting patient fibroblasts with non-modified RNA cocktails, expressing OCT4, SOX2, KLF4, cMYC, NANOG, and LIN28 reprogramming factors. The pluripotency of these patient-derived iPSCs was identified by immunocytochemistry, real-time quantitative polymerase chain reaction, and teratoma formation in vivo in non-obese diabetic/severe combined immunodeficiency mice. RESULTS: All iPSCs derived from patients significantly expressed the pluripotent transcription factors and could be expanded in vitro. Furthermore, induction of terminal differentiation in long-term culture and the capability of forming embryonic bodies to differentiate into all 3 germ layers in vivo were confirmed in immune-deficient mice. CONCLUSION: Fibroblasts from a keloid patient were successfully reprogrammed to iPSCs in vitro. This reprogramming may provide a basis for the production of individualized modified artificial skin to prevent rejections after xenogeneic skin transplantation and trauma through autologous skin transplantation. These cells can also offer a new platform for research on mechanisms underlying skin diseases and personal medical applications.

[Distribution and drug resistance of pathogens at hematology department of Jiangsu Province from 2014 to 2015: results from a multicenter, retrospective study].

Objective: To describe the distribution and drug resistance of pathogens at hematology department of Jiangsu Province from 2014 to 2015 to provide reference for empirical anti-infection treatment. Methods: Pathogens were from hematology department of 26 tertiary hospitals in Jiangsu Province from 2014 to 2015. Antimicrobial susceptibility testing was carried out according to a unified protocol using Kirby-Bauer method or agar dilution method. Collection of drug susceptibility results and corresponding patient data were analyzed. Results: The separated pathogens amounted to 4 306. Gram-negative bacteria accounted for 64.26%, while the proportions of gram-positive bacteria and funguses were 26.99% and 8.75% respectively. Common gram-negative bacteria were Escherichia coli (20.48%) , Klebsiella pneumonia (15.40%) , Pseudomonas aeruginosa (8.50%) , Acinetobacter baumannii (5.04%) and Stenotropho-monas maltophilia (3.41%) respectively. CRE amounted to 123 (6.68%) . Common gram-positive bacteria were Staphylococcus aureus (4.92%) , Staphylococcus hominis (4.88%) and Staphylococcus epidermidis (4.71%) respectively. Candida albicans were the main fungus which accounted for 5.43%. The rates of Escherichia coli and Klebsiella pneumonia resistant to carbapenems were 3.5%-6.1% and 5.0%-6.3% respectively. The rates of Pseudomonas aeruginosa resistant to tobramycin and amikacin were 3.2% and 3.3% respectively. The resistant rates of Acinetobacter baumannii towards tobramycin and cefoperazone/sulbactam were both 19.2%. The rates of Stenotrophomonas maltophilia resistant to minocycline and sulfamethoxazole were 3.5% and 9.3% respectively. The rates of Staphylococcus aureus, Enterococcus faecium and Enterococcus faecalis resistant wards vancomycin were 0, 6.4% and 1.4% respectively; also, the rates of them resistant to linezolid were 1.2%, 0 and 1.6% respectively; in addition, the rates of them resistant to teicoplanin were 2.8%, 14.3% and 8.0% respectively. Furthermore, MRSA accounted for 39.15% (83/212) . Conclusions: Pathogens were mainly gram-negative bacteria. CRE accounted for 6.68%. The rates of Escherichia coli and Klebsiella pneumonia resistant to carbapenems were lower compared with other antibacterial agents. The rates of gram-positive bacteria resistant to vancomycin, linezolid and teicoplanin were still low. MRSA accounted for 39.15%.

Bioinformatic and expression analysis of the OMT gene family in Pyrus bretschneideri cv. Dangshan Su.

With high nutritional value in its fruits, Dangshan Su pear has been widely cultivated in China. The stone cell content in fruits is a key factor affecting fruit quality in pear, and the formation of stone cells has been associated with lignin biosynthesis. O-Methyltransferase (OMT) is a key enzyme involved in lignin metabolism within the phenylpropanoid pathway. Here, we screened 26 OMT genes from the Pyrus bretschneideri cv. Dangshan Su genome using the DNATOOLs software. To characterize the OMT gene family in pear, gene structure, chromosomal localization, and conserved motifs of PbOMTs were analyzed. PbOMTs were divided into two categories, type I (designated PbCCOMTs) and type II (designated PbCOMTs), indicating the differentiation of function during evolution. Based on the analysis of multiple sequence alignment, cis-element prediction, and phylogenetic relationships, two candidate genes, PbCCOMT1 and PbCCOMT3, were selected for the analysis of temporal and spatial gene expression in pear. The promoter regions of both PbCCOMT1 and PbCCOMT3 contain regulatory motifs for lignin synthesis. Moreover, the two genes show high similarity and close phylogenetic relationships with CCOMTs in other species. Expression analysis showed that transcript levels of two PbCCOMTs were positively associated with the contents of both stone cells and lignin during the development of pear fruit. These results suggest that PbCCOMT1 and PbCCOMT3 are closely associated with lignin biosynthesis. These findings will help clarify the function of PbOMTs in lignin metabolism and to elucidate the mechanisms underlying stone cell formation in pear.

Development of a fuzzy-stochastic programming with Green Z-score criterion method for planning water resources systems with a trading mechanism.

This study developed a fuzzy-stochastic programming with Green Z-score criterion (FSGZ) method for water resources allocation and water quality management with a trading-mechanism (WAQT) under uncertainties. FSGZ can handle uncertainties expressed as probability distributions, and it can also quantify objective/subjective fuzziness in the decision-making process. Risk-averse attitudes and robustness coefficient are joined to express the relationship between the expected target and outcome under various risk preferences of decision makers and systemic robustness. The developed method is applied to a real-world case of WAQT in the Kaidu-Kongque River Basin in northwest China, where an effective mechanism (e.g., market trading) to simultaneously confront severely diminished water availability and degraded water quality is required. Results of water transaction amounts, water allocation patterns, pollution mitigation schemes, and system benefits under various scenarios are analyzed, which indicate that a trading-mechanism is a more sustainable method to manage water-environment crisis in the study region. Additionally, consideration of anthropogenic (e.g., a risk-averse attitude) and systemic factors (e.g., the robustness coefficient) can support the generation of a robust plan associated with risk control for WAQT when uncertainty is present. These findings assist local policy and decision makers to gain insights into water-environment capacity planning to balance the basin's social and economic growth with protecting the region's ecosystems.

[Significance of SATB2 in the pathologic diagnosis of osteosarcoma].

OBJECTIVE: To investigate the role of SATB2 in the pathological diagnosis and differential diagnosis of osteosarcoma. METHODS: Immunostaining of SATB2 was performed in 47 cases of osteosarcomas, 5 osteoblastomas, 4 fibrous dysplasias, 5 myositis ossificans, 10 chondroblastomas, 8 chondrosarcomas, 5 Ewing sarcomas, 5 undifferentiated pleomorphic sarcomas, 6 fibrosarcomas and 2 leiomyosarcomas. RESULTS: All osteoblastomas (5/5) and myositis ossificans (5/5), 83.0%(39/47) of osteosarcomas and 2/10 of chondroblastomas showed nuclear immunoreactivity for SATB2. SATB2 staining was negative in all cases of fibrous dysplasia, chondrosarcomas, Ewing sarcomas and all bone primary spindle cell sarcomas(undifferentiated pleomorphic sarcoma, fibrosarcoma and leiomyosarcoma). CONCLUSION: SATB2 is a reliable osteoblastic marker for differential diagnosis of osteosarcoma and non-osteoid sarcoma, although with a limited role in separating osteosarcoma from non-malignant osteoblastic lesions.

µSR investigation of a new diluted magnetic semiconductor Li(Zn,Mn,Cu)As with Mn and Cu codoping at the same Zn sites.

We report the successful synthesis and characterization of a new type I-II-V bulk form diluted magnetic semiconductor (DMS) Li(Zn,Mn,Cu)As, in which charge and spin doping are decoupled via (Cu,Zn) and (Mn,Zn) substitution at the same Zn sites. Ferromagnetic transition temperature up to ∼33 K has been observed with a coercive field ∼40 Oe for the 12.5% doping level. µSR measurements confirmed that the magnetic volume fraction reaches nearly 100% at 2 K, and the mechanism responsible for the ferromagnetic interaction in this system is the same as other bulk form DMSs.

Molecular cloning and characterization of a subtilisin-like protease from Arabidopsis thaliana.

The Arabidopsis thaliana genome encodes 56 subtilisin-like serine proteases (subtilases). In order to evaluate the protease activity of a previously uncharacterized subtilase, designated as AtSBT1.9, we cloned its full-length cDNA from A. thaliana seedlings. An AtSBT1.9 mature peptide coding sequence was inserted into the bacterial expression vector, pMAL-c2x, and the recombinant vector was transformed into Escherichia coli BL21 (DE3). The recombinant AtSBT1.9 tagged by maltose binding protein (MBP) was induced as a 117.5-kDa protein in the soluble form in E. coli BL21 (DE3). MBP-AtSBT1.9 was expressed at a level of 11% (w/w) of the bacterial total protein. Protein purification using Amylose Resin revealed a recombinant AtSBT1.9 protease activity of 9.23 U/mg protein at pH 7 and 25°C. Maximal activity occurred over a broad pH (7-8) and temperature (25°-42°C) optimal range. Validation of AtSBT1.9 protease activity would help in characterizing its in vivo function in A. thaliana.

Orbital characters and electronic correlations in KCo2Se2.

We report a comprehensive study of the tridimensional nature and orbital characters of the low-energy electronic structure in KCo2Se2, using polarization- and photon energy-dependent angle-resolved photoemission spectroscopy. We observed one electron-like Fermi surface (FS) at the Brillouin zone (BZ) center, four electron-like FSs centered at the BZ corner, and one hole-like FS at the BZ boundary. The FSs show weak dispersion along the kz direction, indicating the near-two-dimensional nature of FSs in KCo2Se2. In combination with the local-density approximation calculations, we determined the orbital characters of the low-energy electronic bands, which are mainly derived from the Co 3d orbital, mixed with part of the Se 4p states. The [Formula: see text] orbital gives a significant contribution to the band crossing the Fermi level. A band renormalization of about 1.6 is needed to capture the essential dispersive features, which suggests that electronic correlations are much weaker than that in KyFe2-xSe2.

An integrated simulation and optimization approach for managing human health risks of atmospheric pollutants by coal-fired power plants.

UNLABELLED: This research developed a simulation-aided nonlinear programming model (SNPM). This model incorporated the consideration of pollutant dispersion modeling, and the management of coal blending and the related human health risks within a general modeling framework In SNPM, the simulation effort (i.e., California puff [CALPUFF]) was used to forecast the fate of air pollutants for quantifying the health risk under various conditions, while the optimization studies were to identify the optimal coal blending strategies from a number of alternatives. To solve the model, a surrogate-based indirect search approach was proposed, where the support vector regression (SVR) was used to create a set of easy-to-use and rapid-response surrogates for identifying the function relationships between coal-blending operating conditions and health risks. Through replacing the CALPUFF and the corresponding hazard quotient equation with the surrogates, the computation efficiency could be improved. The developed SNPM was applied to minimize the human health risk associated with air pollutants discharged from Gaojing and Shijingshan power plants in the west of Beijing. Solution results indicated that it could be used for reducing the health risk of the public in the vicinity of the two power plants, identifying desired coal blending strategies for decision makers, and considering a proper balance between coal purchase cost and human health risk. IMPLICATIONS: A simulation-aided nonlinear programming model (SNPM) is developed. It integrates the advantages of CALPUFF and nonlinear programming model. To solve the model, a surrogate-based indirect search approach based on the combination of support vector regression and genetic algorithm is proposed. SNPM is applied to reduce the health risk caused by air pollutants discharged from Gaojing and Shijingshan power plants in the west of Beijing. Solution results indicate that it is useful for generating coal blending schemes, reducing the health risk of the public, reflecting the trade-offbetween coal purchase cost and health risk.

Waste management with recourse: an inexact dynamic programming model containing fuzzy boundary intervals in objectives and constraints.

The existing inexact optimization methods based on interval-parameter linear programming can hardly address problems where coefficients in objective functions are subject to dual uncertainties. In this study, a superiority-inferiority-based inexact fuzzy two-stage mixed-integer linear programming (SI-IFTMILP) model was developed for supporting municipal solid waste management under uncertainty. The developed SI-IFTMILP approach is capable of tackling dual uncertainties presented as fuzzy boundary intervals (FuBIs) in not only constraints, but also objective functions. Uncertainties expressed as a combination of intervals and random variables could also be explicitly reflected. An algorithm with high computational efficiency was provided to solve SI-IFTMILP. SI-IFTMILP was then applied to a long-term waste management case to demonstrate its applicability. Useful interval solutions were obtained. SI-IFTMILP could help generate dynamic facility-expansion and waste-allocation plans, as well as provide corrective actions when anticipated waste management plans are violated. It could also greatly reduce system-violation risk and enhance system robustness through examining two sets of penalties resulting from variations in fuzziness and randomness. Moreover, four possible alternative models were formulated to solve the same problem; solutions from them were then compared with those from SI-IFTMILP. The results indicate that SI-IFTMILP could provide more reliable solutions than the alternatives.

[Selective isolation of anethole from volatile oil of Foeniculum vulgare Mill by inclusion crystalline with chela-shape host].

AIM: To isolate the components from the volatile oil of Foeniculum vulgare Mill. METHODS: According to the function of molecular recognition of supramolecular chemistry, chela shape molecule, trans-1, 2-biphenyl-1, 2-acenaphthendiol was used as host molecule and the volatile oil of Foeniculum vulgare Mill as guest molecule. Trans-1, 2-biphenyl-1, 2-acenaphthendiol can recognize the components that endowed with interactional complementarity and form inclusion compound as crystals. RESULTS: The anethole in the volatile oil was selectively included as trans-1,2-biphenyl-1,2-acenaphthendiol which was obtained in pure state from the inclusion compound by Kugelrohr vacuum technology. The formation of inclusion compound was confirmed by means of IR and powder XRD. The structure of the selectively isolated component was elucidated as trans-anethole by means of IR, 1HMMR and MS. CONCLUSION: The experimental results showed that the method is simple, rapid and selective for isolation anethole from volatile oil of Foeniculum vulgare Mill.

Characterization of epitopes for virus-neutralizing monoclonal antibodies to Ross River virus E2 using phage-displayed random peptide libraries.

Ross River virus (RRV) is the predominant cause of epidemic polyarthritis in Australia, yet the antigenic determinants are not well defined. We aimed to characterize epitope(s) on RRV-E2 for a panel of monoclonal antibodies (MAbs) that recognize overlapping conformational epitopes on the E2 envelope protein of RRV and that neutralize virus infection of cells in vitro. Phage-displayed random peptide libraries were probed with the MAbs T1E7, NB3C4, and T10C9 using solution-phase and solid-phase biopanning methods. The peptides VSIFPPA and KTAISPT were selected 15 and 6 times, respectively, by all three of the MAbs using solution-phase biopanning. The peptide LRLPPAP was selected 8 times by NB3C4 using solid-phase biopanning; this peptide shares a trio of amino acids with the peptide VSIFPPA. Phage that expressed the peptides VSIFPPA and LRLPPAP were reactive with T1E7 and/or NB3C4, and phage that expressed the peptides VSIFPPA, LRLPPAP, and KTAISPT partially inhibited the reactivity of T1E7 with RRV. The selected peptides resemble regions of RRV-E2 adjacent to sites mutated in neutralization escape variants of RRV derived by culture in the presence of these MAbs (E2 210-219 and 238-245) and an additional region of E2 172-182. Together these sites represent a conformational epitope of E2 that is informative of cellular contact sites on RRV.

Tris(2-cyanoethyl)amine.

In the title compound, N(CH(2)CH(2)CN)(3), (I), the three cyanoethyl groups adopt a conformation with the CN groups oriented in the same direction, suggesting the compound may behave as a potential tripodal ligand.

Multiple alignment and sorting of peptides derived from phage-displayed random peptide libraries with polyclonal sera allows discrimination of relevant phagotopes.

Biopanning of phage-displayed random peptide libraries is a powerful technique for identifying peptides that mimic epitopes (mimotopes) for monoclonal antibodies (mAbs). However, peptides derived using polyclonal antisera may represent epitopes for a diverse range of antibodies. Hence following screening of phage libraries with polyclonal antisera, including autoimmune disease sera, a procedure is required to distinguish relevant from irrelevant phagotopes. We therefore applied the multiple sequence alignment algorithm PILEUP together with a matrix for scoring amino acid substitutions based on physicochemical properties to generate guide trees depicting relatedness of selected peptides. A random heptapeptide library was biopanned nine times using no selecting antibodies, immunoglobulin G (IgG) from sera of subjects with autoimmune diseases (primary biliary cirrhosis (PBC) and type 1 diabetes) and three murine ascites fluids that contained mAbs to overlapping epitope(s) on the Ross River Virus envelope protein 2. Peptides randomly sampled from the library were distributed throughout the guide tree of the total set of peptides whilst many of the peptides derived in the absence of selecting antibody aligned to a single cluster. Moreover peptides selected by different sources of IgG aligned to separate clusters, each with a different amino acid motif. These alignments were validated by testing all of the 53 phagotopes derived using IgG from PBC sera for reactivity by capture ELISA with antibodies affinity purified on the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), the major autoantigen in PBC: only those phagotopes that aligned to PBC-associated clusters were reactive. Hence the multiple sequence alignment procedure discriminates relevant from irrelevant phagotopes and thus a major difficulty with biopanning phage-displayed random peptide libraries with polyclonal antibodies is surmounted.